3. MATERIALS AND METHODS
3.7 Analytical methods
3.7.1Assay of cutinase
The activity was measured by following the hydrolysis of p-nitrophenyl butyrate (p-NPB) (Sigma) as substrate. An aliquot of (0.020 ml) culture supernatant was added to 0.98 ml of reaction mixture, which was prepared by adding 1 ml of 23 mM pNPB in tetrahydrofuran to 40 ml of 50 mM potassium phosphate buffer containing 11.5 mM of sodium deoxycholate.
The reaction was monitored for 15 minutes at 37ºC and absorbance of released p-nitrophenol measured at 410 nm. Activity with cutinase specific substrate, p-nitrophenyl (16 methyl sulphone ester) hexadecanoate (p-NMSH) was measured by method described by Degani et al., (2006). An aliquot of (0.1 ml) culture supernatant was added to 0.9 ml of reaction mixture, which was prepared by adding 1 ml of 23 mM pNMSH in tetrahydrofuran to 40 ml of 50 mM potassium phosphate buffer of pH 8. The enzyme substrate mixture was incubated for 4 h at 37ºC and absorbance of released p-nitrophenol measured as above. One enzyme unit is defined as the amount of enzyme required to release one µMol of p-nitrophenol min-1 under assay conditions. The method for preparation of p-nitrophenol standard curve was described in appendix A.2.
3.7.2 Protein determination
The total protein content of the samples was determined according to the method described by Lowry et al., (1951). The protein assay mixture consisted of 200 µl of diluted crude extract of enzyme (5 times) and 1 ml of freshly prepared complex forming reagent (2%
Na2CO3 in 0.1 N NaOH : 1.0% CuSO4.5H2O : 2% potassium sodium tartarate ≡ 100:1:1).
The contents in the sample were vortexed and incubated at room temperature for 15 min.
Then 100 µ l of freshly prepared diluted Folin reagent with distilled water (1:1) was added
and vortexed and kept in the dark at room temperature for 30 min. Blue color was developed and OD at 660 nm was measured against the blank (no crude enzyme, 200 µl distilled water). The protein concentration in the reaction was determined based on a standard curve (appendix A.3) obtained with bovine serum albumin (Sigma) as standard.
3.7.3 Dry cell weight (DCW)
Due to the presence of cutin, cell dry-weight concentration could not be measured directly and therefore, intracellular protein concentration was measured (Agarwal et al., 2009). For standard curve of DCW, cells were centrifuged at 10,000g for 10 min at 4±1ºC and separated from the supernatant. The pellet was resuspended with same volume of distilled water and centrifuged at 10,000g for 10 min at 4±1ºC, washed twice with distilled water and supernatant free cells were used to determine the DCW. Different dilutions of cell samples were used for measuring intracellular protein concentration. For measuring intracellular protein, cells were washed as above and subjected to lysis by dissolving the cell samples in 1ml of 0.4 N NaOH. Then the samples were heated at 100ºC for 20 min. After cooling down the sample at room temperature, the protein concentration was measured using method described in section 3.7.2. DCW of the unknown sample was determined by measuring the intracellular protein concentration and compared with standard curve (appendix A.4) between dry cell weight and intracellular protein (Cell dry weight in g l-1 = Intracellular protein in g l-1 × 19.03).
3.7.4 Estimation of alkyl ester
The ester formation was estimated by consumption of fatty acid. To estimate the fatty acid consumption, 300 µl of the mixture (for butyric acid) or 100 µl (for all other acids) sample
were transferred to 2 ml micro centrifuge tube and centrifuged at 10,000g for 1 min to remove the suspended enzyme particles. The progress of reaction was determined by measuring the decreasing profile of acids by titration with 0.01 N NaOH using phenolphthanein as indicator and also with Lowry and Tinsley method (1976). According to Lowry and Tinsley method, the samples were diluted (7:3 for butyric acid or 9:1 for other) in isooctane making the total volume of 1 ml. Cupric acetate aqueous solution (0.2 ml) containing pyridine (5%, w/v, pH 6.0) was then added into the tube and the solutions were vigorously mixed for 1 min using a vortex mixture. After centrifugation at 5000g for 5 min, the upper organic phase was measured by a UV/visible spectrophotometer (Cary 100, Varian) at 715 nm. The amount of fatty acid consumed was calculated using the standard curve for that fatty acid (Appendix A.5).
3.7.5 GC analysis of esters
Synthesis of fatty acid ester was analyzed by gas chromatograph (Varian 390). The diluted aliquots of the reaction mixture were injected into CPSIL 8CB column and compounds were detected by FID. The injector and detector temperature were set at 250°C. The program (temperature and time) and retention time (tR) used for different esters are given below.
Butyl butyrate: 150°C for (0.5min) -15 °C/min-250°C (10min); tR 2.42min Butyl valerate: 150°C for (0.5min) -15 °C/min-250°C (10min); tR 3.14 min Butyl hexanoate: 150°C for (0.5min) -15 °C/min-250°C (10min); tR 4.38 min Butyl octanoate: 150°C for (0.5min) -30 °C/min-250°C (10min); tR 6.65 min Butyl decanoate: 150°C for (0.5min) -30 °C/min-270°C (10min); tR 15.5 min Butyl Palmitate: 150°C for (0.5min) -30 °C/min-300°C (10min); tR 20.3 min Ethyl butyrate: 100°C for (0.5min) -15 °C/min-150°C (10min); tR 1.36min
Ethyl valerate: 100°C for (0.5min) -15 °C/min-150°C (10min); tR 2.55 min Ethyl hexanoate: 50°C for (0.5min) -30°C/min-160°C (10min); tR 2.15 min Pentyl butyrate: 150°C for (0.5min) -30 °C/min-250°C (10min); tR 3.58 min Hexyl butyrate: 150°C for (0.5min) -30 °C/min-250°C (10min); tR 4.18 min Octyl Butyrate: 150°C for (0.5min) -30 °C/min-250°C (10min); tR 5.43 Decyl butyrate: 100°C for (0.5min) -30 °C/min-150°C (10min); tR 2.20 min Methyl butyrate: 150°C for (0.5min) -15 °C/min-250°C (10min); tR 1.69min Methyl palmitate: 150°C for (0.5min) -30 °C/min-250°C (10min); tR 3.14 min Methyl oleate: 150°C for (0.5min) -30 °C/min-250°C (10min); tR 4.88 min Methyl linoleate: 150°C for (0.5min) -30 °C/min-250°C (10min); tR 2.67 min